Cigarette smoking remains a major global health burden and is strongly associated with increased childhood respiratory infections and development of chronic lung diseases such as COPD, asthma and lung cancer. The adverse health effects of cigarette smoke (CS) are largely related to its immuno-suppressive properties leading to impaired innate immune responses, host defense, and tumor surveillance. Although it is commonly thought that these effects are due to CS-derived reactive oxygen species (ROS), largely based on observations of protective effects of thiol-based antioxidants, the main thiol-reactive agents within CS are acrolein (2,3- propenal) and other related unsaturated aldehydes. Our recent studies in mice have demonstrated immunosuppressive effects of acrolein on alveolar macrophages that mimic those of CS. Acute mechanisms associated with direct and transient alkylation of redox-sensitive protein targets appear to be critical in this respect, diretly affecting activation of NF-?B or AP-1 and altering cellular redox regulation. However, the specific functional consequences of these protein alkylations are not known. Acrolein exposure also mimics some of the variable effects of cigarette smoking on asthma development and severity, by promoting allergic sensitization but also by suppressing allergic inflammation. These effects of acrolein were associated with increased epithelial injury and are likely mediated by altered epithelial barrier integrity and interaction with dendritic cells, the main antigen-presentng cell in the lung, although the mechanisms by which acrolein impacts on epithelial integrity and production of mediators that regulate dendritic cell maturation are not known. The main goal of the present proposal is to elucidate the mechanisms by which acrolein alters innate macrophage and epithelial immune responses as well as allergic inflammation, and to identify the importance of direct alkylation of critical enzyme systems. We plan to determine the importance of direct protein modifications in acrolein-induced suppression of innate immune responses (Aim 1), using newly developed proteomic approaches and analysis of the functional importance of these modifications in target proteins, and explore the consequences of acrolein exposure on allergic inflammation and sensitization (Aim 2), focusing on alterations in epithelial barrier function and mediator production in association with direct alkylation or relevant target proteins. Also, based on recent studies indicating that acrolein is detoxified by glutathione S-transferase P1 (GSTP1) and by hydrogen sulfide (H2S), a newly recognized endogenous mediator produced by (homo)cysteine metabolism by cystathionine -synthase (CBS), we will explore the importance of GSTP1 and CBS/H2S in modulating acrolein-induced alterations in immune responses (Aim 3). Collectively, these studies will not only offer important insights into the potential contribution of acrolein to CS-related disease, but may also be relevant to understanding the actions of other relevant endogenous or environmental electrophiles. PUBLIC HEALTH RELEVANCE: Many lines of evidence indicate that the health effects of cigarette smoking are related to alterations of the immune system. While cigarette smoking is generally associated with chronic inflammation, it also possesses immunosuppressive properties that result in increased sensitivity to respiratory bacterial or viral infection. Moreovr, cigarette smoking also variably contributes to allergic asthma, by promoting allergic sensitization but also altering inflammation altering the development or severity of allergic airway disease. The mechanistic aspects of such effects of smoking are poorly understood. Although it is popular belief that the adverse effects of CS are due to oxidative stress, based on protective effects of thiol-based antioxidants, our studies performed with previous NIH support indicate that acrolein (2,3-propenal), an important reactive component of CS, is responsible for many of the effects of CS due to its reactivity to thiols and redox-sensitive pathways. Moreover, acrolein exposure mimics many of the adverse effects of CS on the immune system, and can similarly affect allergic inflammation. Using proteomics approaches, we have identified the major cell targets for acrolein and show that acrolein directly targets several critical cell proteins involve in inflammatory signaling and immune responses. The main goal of the current proposal is to address the importance for such direct protein modifications in in vivo mouse models of acute lung injury and of allergic asthma. Secondly, we will address the importance of enzymatic and metabolic systems that mediate detoxification of acrolein, to determine whether changes in these systems alter the susceptibility to acrolein. Collectively, these studies will provide important insights into the importance of acrolein in development of respiratory disease associated with smoking or other environmental exposure, and will help determine factors that may contribute to susceptibility to CS-induced respiratory infections or allergic airways disease.